Research efforts focus on vaccine and therapeutic development through both basic and applied research projects. Studies are designed to increase our understanding of pathogenic mechanisms associated with microbial infections and devise novel vaccine and therapeutics strategies to protect humans from the severe effects of infectious diseases. In recent research studies, our laboratory determined the molecular mechanism responsible for antibiotic resistance in several clinical isolates of Streptococcus pneumoniae from patients at Children's Hospital in Washington, DC. In one study, we demonstrated that a specific single amino acid mutation in the bacterial chromosomal gene for dihydrofolate reductase was responsible for high-level trimethoprim resistance seen in these pneumococcal clinical isolates. Using a computer model, protein crystalization data, and site-directed mutagenesis, we solved the molecular mechanism at the atomic level of two angstroms. In a similar study, we have isolated and characterized the molecular determinant responsible for optochin resistance in S. pneumoniae. This is the first reported case of infection with S. pneumoniae in the United States that cannot be diagnosed by the optochin sensitivity disc test. We have isolated the gene and the mutation responsible for failure of this strain to be detected by the clinical laboratory test and have now developed a molecular model that explains the resistance. In addition to this research, the laboratory continues to work on the development of genetically detoxified pertussis toxin for acellular whooping cough vaccines. Whooping cough is an upper respiratory tract infection caused by Bordetella pertussis resulting in mortality rates estimated at about 500,000 deaths per year. This disease has been effectively controlled by the current vaccine which consists of killed whole B. pertussis cells. Although efficacious, the present whole cell vaccine produces unacceptable side effects. The major protective antigen in whooping cough vaccines is pertussis toxin. Chemically inactivated pertussis toxin vaccines have been produced with reduced side effects and reasonable efficacy, however, these products suffer from reduced antigenicity and difficulties in vaccine production processing. In addition, residual activity may exist from reversion of the chemical treatment or incomplete chemical inactivation. Using site-specific DNA mutagenesis, we modified E. coli subclones of pertussis toxin and used these constructs to replace the chromosomal copy of the toxin gene in B. pertussis vaccine strain 3779. The resulting new strain produces a fully genetically detoxified form of pertussis toxin that is strongly immunoprotective and can be used as a vaccine antigen without chemical inactivation. In a recently completed NIAID-supported clinical trial in Sweden and Italy, pertussis toxin emerged as an essential component of any new whooping cough vaccine. One of the most successful acellular pertussis vaccines used in this clinical trial contained a genetically altered version of pertussis toxin that was developed from basic research generated through this intramural research project. Molecular studies are currently underway in our laboratory to develop higher yield bacterial strains to enhance expression of pertussis toxin for use in acellular and conjugate vaccine production. In addition to this work, we have begun a program to develop new generation, plant derived vaccines. This system uses both transgenic plants and a transient expression viral system to produce immunoprotective protein antigens from organisms such as Vibrio cholerae, Mycobacterium tuberculosis, Bacillus anthracis, Streptococcus mutans and hepatitis C virus. An experimental HCV vaccine has been produced in plants and is currently being tested.